WO1990002557A1 - Vaccines and diagnostic assays for haemophilus influenzae - Google Patents

Vaccines and diagnostic assays for haemophilus influenzae Download PDF

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Publication number
WO1990002557A1
WO1990002557A1 PCT/US1989/003779 US8903779W WO9002557A1 WO 1990002557 A1 WO1990002557 A1 WO 1990002557A1 US 8903779 W US8903779 W US 8903779W WO 9002557 A1 WO9002557 A1 WO 9002557A1
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Prior art keywords
pbomp
protein
amino acid
peptide
dna
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PCT/US1989/003779
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English (en)
French (fr)
Inventor
Algis Anilionis
Robert C. Seid, Jr.
Robert A. Deich
Gary W. Zlotnick
Bruce A. Green
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Praxis Biologics, Inc.
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Priority claimed from US07/239,572 external-priority patent/US5098997A/en
Application filed by Praxis Biologics, Inc. filed Critical Praxis Biologics, Inc.
Priority to EP89910798A priority Critical patent/EP0432220B1/de
Priority to KR1019980702574A priority patent/KR0170752B1/ko
Priority to DE68923286T priority patent/DE68923286T2/de
Publication of WO1990002557A1 publication Critical patent/WO1990002557A1/en
Priority to DK199100358A priority patent/DK174965B1/da

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the present invention relates to compositions and methods for the preparation of proteins and peptides
  • the invention is directed to compositions and methods for preparation of proteins and peptides related to a class of outer membrane proteins of about 16000 daltons molecular weight of type b and non-typable H. influenzae including PBOMP-1 and PBOMP-2.
  • the proteins and peptides are used as immunogens in vaccine formulations for active
  • the proteins and peptides can be obtained by novel improved methods of purification from H. influenzae or produced using either recombinant DNA or chemical synthetic methods. Additionally, the invention relates to novel DNA sequences and vectors useful for directing expression of PBOMP-1 and PBOMP-2 related proteins and peptides. Thenucleotide sequences are used as reagents in nucleic acid hybridization assays.
  • Recombinant DNA technology involves insertion of specific DNA sequences into a DNA vehicle (vector) to form a recombinant DNA molecule which is capable of replication in a host cell.
  • the inserted DNA sequence is foreignto the recipient DNA vehicle, i.e., the inserted DNA sequence and the DNA vector are derived from organisms which do not exchange genetic information in nature, or the inserted DNA sequence may be wholly or partially synthetically made.
  • Pat. No. 4,237,224 to Cohen and Boyer describes production of such recombinant plasmids using processes of cleavage with restriction enzymes and joining with DNA ligase by known methods of ligation. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. Because of the general applicability of the techniques described therein,
  • This method utilizes a packagmg/transduction system with bacteriophage vectors
  • Recombinant genes may also be introduced into viruses, such as vaccina virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • the recombinant DNA molecule must be compatible with the host cell, i.e., capable of autonomous replication in the host cell or stably integrated into one of the host cell's chromosomes.
  • the recombinant DNA molecule or virus e.g., a vaccinia virus recombinant
  • virus es
  • the foreign gene will be properly expressed in the transformed bacterial cells, as is the case with bacterial expression plasmids, or in permissive cell lines infected with a recombinant virus or a recombinant plasmid carrying a eucaryotic origin of replication.
  • Different genetic signals and processing events control many levels of gene expression; for instance, DNA
  • mRNA messenger RNA
  • eucaryotic promotors Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eucaryotic promotors differ from those of procaryotic promotors.
  • eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system and further, procaryotic promotors are not recognized and do not function in
  • SD Shine-Dalgarno
  • hybrid genes in which the foreign sequence is ligated in phase (i.e., in the correct reading frame) with a procaryotic gene. Expression of this hybrid gene results in a fusion protein product (a protein that is a hybrid of procaryotic and foreign amino acid sequences).
  • Expression vectors have been used to express genes in a suitable host and to increase protein production.
  • the cloned gene should be placed next to a strong promotor which is controllable so 'that transcription can be turned on whan necessary.
  • Cells can be grown to a high density and then the promotor can be induced to increase the number of
  • transcripts These, if efficiently translated will result in high yields of protein. This is an especially valuable system if the foreign protein is deleterious to the host cell.
  • ColEl-type replicons Most plasmid cloning vectors commonly used in E. coli are derivatives of ColEl-type replicons (for additional information see Oka et al., 1979, Mol. Gen. Genet. 172:151- 159).
  • the ColEl plasmids are stably maintained in E. coli strains as monomeric molecules with a copy number of about
  • One way to obtain large amounts of a given gene product is to clone a gene on a plasmid which has a very high copy number within the bacterial cell.
  • mRNA levels should also increase which should lead to increased production of the recombinant protein.
  • Vaccinia virus may be used as a cloning and expression vector.
  • the virus contai.ns a li.near double-stranded DNA genome of approximately 187 kb pairs which replicates within the cytoplasm of infected cells.
  • These viruses contain a complete transcriptional enzyme system (including capping, methylating and polyadenylating enzymes) within the virus core which are necessary for virus infectivity.
  • Vaccinia virus transcriptional regulatory sequences promotors allow for initiation of transcription by vaccinia RNA polymerase but not by eucaryotic RNA polymerase.
  • Plasmid vectors also called insertion vectors have been constructed to insert the
  • insertion vector is composed of: (1) a vaccinia virus promotor
  • transcri.ptional initiation site including the transcri.ptional initiation site; (2) several unique restriction endonuclease cloning sites downstream from the transcriptional start site for insertion of foreign DNA fragments; (3) nonessential vaccinia virus DNA (such as the
  • TK gene flanking the promotor and cloning sites which direct insertion of the chimeric gene into the homologous
  • Recombinant viruses are produced by transfection of recombinant bacterial insertion plasmids containing the foreign gene into cells infected with vaccinia virus.
  • vaccinia recombinants retain their essential functions and infectivity and can be constructed to accommodate approximately 35 kb of foreign DNA.
  • enzymatic or immunological assays e.g., immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, or immunoblotting.
  • ELISA enzyme-linked immunosorbent assay
  • radioimmunoassay radioimmunoassay
  • immunoblotting naturally occurring membrane glycoproteins produced from recombinant vaccinia infected cells are glycosylated and may be transported to the cell surface. High expression levels can be obtained by using strong promotors or cloning multiple copies of a single gene in appropriate vectors and suitable hosts.
  • a baculovirus such as Autographica californica nuclear polyhedrosis virus (AcNPV) may also be used as a cloning or expression vector.
  • the infectious form of AcNPV is normally found in a viral occlusion. This structure is largely composed of polyhedrin peptide in which virus particles are embedded. Polyhedrin gene expression occurs very late in the infection cycle, after mature virus particles are formed.
  • polyhedrin gene expression is a dispensible
  • non-occluded virus particles produced in the absence of polyhedrin gene expression are fully active and are capable of infecting cells in culture.
  • a recombinant baculovirus expression vector is prepared by cleaving baculovirus DNA to produce a fragment comprising a polyhedrin gene or portion thereof, inserting this fragment into a cloning vehicle and thereafter inserting the gene to be expressed such that it is under control of the polyhedrin gene promotor.
  • the recombinant transfer vector formed in this way is mixed with baculovirus helper DNA and used to transfect insect cells in culture to effect recombination and incorporation of the selected gene at the polyhedrin gene locus of the baculovirus genome.
  • the resultant recombinant baculovirus is used to infect susceptible insects or cultured insect cells.
  • H. influenzae are divided into two groups. Those strains which possess a known capsule are typed by the serological reaction of the capsule with reference antisera. Types a-f have been identified. Strains which fail to react with any of the reference antisera are known as non-typable.
  • H. influenzae type b (Hib) is the most frequent cause of neonatal meningitis and other invasive infections in the Unites States (Fraser et al., 1974, Am. J. Epidemiol.
  • influenzae also cause diseases including pneumonia, bacteremia, meningitis, postpartum sepsis, and acute febrile tracheobronchitis in adults (Murphy et al., 1985, J. Infect.
  • Non-typable Hi are a frequent etiologic agent of otitis media in children and young adults, causing about 20 to 40% of all otitis media cases. Children may experience multiple infections due to the same organism since infection confers no long lasting immunity. Current therapy for chronic or repeated occurrences of otitis media includes administration of antibiotics and insertion of tubes to drain the inner ear. Hi strains have also been implicated as a primary cause of sinusitis (Cherry J.D. and J.P. Dudley,
  • Hib H. influenzae
  • PRP polyribosyl ribitol phosphate
  • Anti-PRP antibody is ineffective against non-typable H.
  • the ideal candidate for a Haemophilus vaccine would have three properties: a) it would be immunogenic in infants of 2-6 months (b) it would elicit an antibody which would protect against infections caused by typable and non-typable H. influenzae, and (c) it would el ⁇ cit antibody against a determinant found on the surface of all strains of H.
  • Hib infections consist essentially of PRP, the type b
  • PRP polysaccharide Purified PRP polysaccharide is immunogenic in children above 18 months of age, but does not elicit a protective antibody response in those younger than 18 months. In general, polysaccharides have been shown to be poor immunogens in children less than about 18 months of age.
  • formulations address one difficulty of PRP vaccines, i.e., their inability to protect infants younger than 18 months, they fail to address another major problem of the PRP
  • Anti-PRP antibody is ineffective against non- typable H. influenzae, which by definition lack the PRP capsule. Hence there is a long recognized need for a vaccine that will elicit a protective immune response in children of about 18 months and younger against both typable, including type b and non-typable H. influenzae.
  • One object of the present invention is to provide a vaccine formulation that elicits a protective immune response against typable H. influenzae including type b and non- typable H. influenzae in children under 6 months as well as in older children and adults.
  • the approach of the present invention is to vaccinate with a protein or fragment thereof which is exposed on the surface of Haemophilus.
  • the best candidate is an outer membrane protein (OMP) of H.
  • Outer membrane proteins are usually surface exposed molecules. They are composed of protein which is normally immunogenic in infants, and they have been shown to be capable of eliciting protective antibody in other
  • Antibody to an OMP of Haemophilus could be both bactericidal and opsonic much as anti-PRP has been shown to be bactericidal and opsonic for Hib (Anderson et al., 1972,
  • the present invention is directed to peptides and proteins related to an outer membrane protein of about 16000 daltons molecular weight of Haemophilus influenzae identified by applicants and termed "Praxis Biologies Outer Membrane Protein-1" (PBOMP-1) and to an antigenically related outer membrane protein of about 16000 daltons molecular weight of Haemophilus influenzae also identified by applicants and termed “Praxis Biologies Outer Membrane Protein-2" (PBOMP-2), as well as the molecularly cloned genes or gene fragments whr.ch encode these peptides or proteins.
  • PBOMP-1 an outer membrane protein of about 16000 daltons molecular weight of Haemophilus influenzae identified by applicants and termed "Praxis Biologies Outer Membrane Protein-1" (PBOMP-1)
  • PBOMP-2 Protecte Biologies Outer Membrane Protein-2
  • PBOMP-1 PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein
  • PBOMP-1 fusion protein as well as the molecularly cloned genes or gene fragments which encode these peptides or proteins.
  • chemically synthesized PBOMP-1 or PBMOP-2 related peptides encompass an antigenic region(s) of PBOMP-1 or PBOMP-2 respectively.
  • the peptides are conjugated to a protein carrier, resulting in the generation of an
  • immunogenic peptide conjugate The invention is also
  • influenzae using novel and improved methods.
  • the peptides or proteins of the present invention may be used as immunogens in vaccine formulations for H. influenzae, or as reagents in diagnostic immunoassays for H. influenzae.
  • the present invention is also directed to methods for the molecular cloning of genes or gene fragments encoding PBOMP-1 and PBOMP-2 related peptides. These molecularly cloned sequences can then be used in the further construction of other vectors by recombinant DNA techniques, including expression vectors for the encoded peptide products, or use in diagnostic assays for H. influenzae based on nucleic acid hybridization, or in construction of a sequence encoding a PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein.
  • the peptides or proteins of the present invention may be purified from H. influenzae, or produced using recombinant
  • novel DNA sequences and vectors including plasmid DNA, and viral DNA such as human viruses, animal viruses, insect viruses, or bacteriophages which can be used to direct the expression of PBOMP-1 and
  • PBOMP-2 or PBOMP-2 PBOMP-1 related peptides or proteins in appropriate host cells from which the peptides and proteins may be purified. Chemical methods for the synthesis of
  • PBOMP-1, PBOMP-2, PBOMP-1 PBOMP-2 and PBOMP-2: PBOMP-1 related peptides and proteins can be used as immunogens in subunit vaccine formulations for use against all pathogenic H. influenzae, including both type b and non-typable H.
  • PBOMP-1 and PBOMP-2 related proteins or peptides for subunit vaccine preparations can be obtained by chemical synthesis, purification from H. influenzae or purification from recombinant expression vector systems.
  • PBOMP-1: PBOMP-2 and PBOMP-2: PBOMP-1 related proteins or peptides for subunit vaccine preparations can be obtained by purification from recombinant expression vector systems or by chemical
  • recombinant viruses which produce the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, or PBOMP-2: PBOMP-1 related peptides or proteins themselves or extracts of cells infected with such recombinant viruses can be used as
  • PBOMP-1 or PBOMP-2 protein or PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 fusion protein will be recognized as "foreign" in the host animal, a humoral and possibly a cell-mediated immune
  • PBOMP-1 PBOMP-2 or PBOMP-2: PBOMP-1 respectively. In a properly prepared vaccine formulation, this should protect the host against subsequent H. influenzae infections.
  • present subunit vaccine formulations will be compatible with currently available PRP vaccines.
  • PBOMP-1-related and/or PBOMP-2 related sequences of the present invention can be used in human medical assays.
  • peptides and proteins of the present invention include the use of the peptides and proteins of the present invention as reagents in immunoassays such as ELISA tests and radioimmunoassays which are useful as diagnostic tools for the detection of H. influenzae infection in blood samples, body fluid, tissues, etc.
  • the PBOMP-1 encoding and/or PBOMP-2-encoding gene sequences can be used in DNA-DNA or DNA-RNA hybridization assays for similar diagnostic detection of H. influenzae. Additionally, these reagents will provide a valuable tool in elucidating the mechanism of pathogenesis of H. influenzae.
  • the present invention is directed further to anti- PBOMP-1, anti-PBOMP-2, anti-PBOMP-1: PBOMP-2, and/or anti- PBOMP-2: PBOMP-1 monoclonal antibodies which have uses in passive immunization regimes, and in diagnostic immunoassays.
  • monoclonal antibodies may be generated against PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and/or PBOMP-2: PBOMP-1 related peptides.
  • FIG. 1 represents sodium dodecylsulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of PBOMP-1.
  • Samples and gels were prepared as described in Section 6.1.
  • Lane A contains about 5 ug PBOMP-1.
  • Lane B contains prestained low molecular weight (MW) standards: ovalbumin, alpha- chymotrypsinogen, beta-lactoglobulin, lysozyme, bovine trypsin inhibitor and insulin (A and B chains). Relative MWs [in kilodaltons (kd)] are shown at the side.
  • MW molecular weight
  • FIG. 2 (A and B) represents reactivity of whole cell lysates of E. coli and H. influenzae with polyclonal anti- PBOMP-1 antibody and a monoclonal anti-PBOMP-1 antibody (G1- 1).
  • lysates were reacted with polyclonal anti- PBOMP-1 antibody.
  • Lanes are as follows: (1) E. coli HB101; (2) E. coli JM83; (3) molecular weight standards; (4)
  • FIG. 2B lysates were reacted with monoclonal anti-PBOMP-1 antibody. Lanes are as descr ⁇ bed in FIG. 2A.
  • FIG. 3 represents a restriction map of pGD103, a derivative of pLG339 (see Stoker et al., 1982, Gene 18:335-
  • FIG. 4 (A and B) represents maps of pAA152 which comprises a 4.2 Kb fragment of H. influenzae DNA cloned into pGD103.
  • a gene encoding PBOMP-1 is localized to an 737 bp
  • FIG. 4A is a circular restriction map of pAA152.
  • FIG. 4B illustrates deletion analysis of the inserted fragment of pAA152. The remaining H. influenzae DNA in the deletion derivatives is denoted by black lines. PBOMP phenotype is noted at the right.
  • FIG . 5 represents reactivity of whole cell lysates of
  • Lanes are as follows: (A) monoclonal antibody G1-1; (b) monoclonal antibody G94-3; (C) monoclonal antibody G18-3; (D) monoclonal antibody 25-2; and (E) monoclonal antibody G2-3.
  • FIG. 6 represents autoradiographic analysis of DS410 minicells containing recombinant plasmids pAA130 and pAA152.
  • Lanes represent: (A) DS410 (pAA130); (B) DS410
  • FIG. 7 (A and B) represents maps of pAA130 which comprises a 5.7 Kb fragment of H. influenzae DNA cloned into pGD103.
  • FIG. 7A represents a circular restriction map of pAA130.
  • FIG. 7B represents deletion analysis of the H.
  • FIG. 8 represents reactivity of whole cell lysates of
  • Lanes represent: (A) JM83 containing pAA130; (B) JM83 containing pAA130; (C) JM83; (D) JM83; (E) molecular weight standards as displayed in
  • FIG. 9 represents the DNA sequencing strategy of the
  • ORF major open reading frame
  • FIG. 10 represents the nucleotide sequence of the 737 bp fragment which contains the PBOMP-1 gene.
  • the predicted open reading frame (ORF) is shown by the underlined sequence and the direction of transcription indicated by the
  • FIG. 11 represents the deduced amino acid sequence of
  • N-terminal amino acid of the mature form of the protein N-terminal amino acid of the mature form of the protein.
  • FIG. 12 represents alignment of the partial amino acid sequence of a peptide derived from PBOMP-1 (below) with a portion of the derived amino acid sequence of the PBOMP-1 gene (above). Residues enclosed within boxes represent mismatches.
  • FIG. 13 represents the sequencing strategy of the 789 bp BstEII-XmnI fragment of pAA130 showing the origin,
  • FIG. 14 represents the nucleotide sequence of the 789 bp BstEII-XmnI fragment of pAA130 which contains the PBOMP-2 gene.
  • the predicted ORF is shown by the underlined sequence.
  • the two bases designated "N" represent unknown nucleotides.
  • FIG. 15 represents the deduced ammo acid sequence of
  • FIG. 16 represents a chromatogram, obtained using gas liquid chromatography, of the fatty acids of PBOMP-1.
  • Nonadecanoic acid (C 19) was included as an internal
  • FIG. 17 represents autoradiographic SDS-PAGE analysis of E. coli JM83 cells containing recombi.nant plasmids pAA130 and pAA152 as well as control E. coli JM83 cells containing pGD103. Lanes represent: (1) pAA130; (2) pAA152 and (3) pGD103. The location of a band of about 15,000 daltons molecular weight is noted at the left of the figure.
  • FIG. 18 (A and B) represents Western blot gel analysis of whole cell lysates of E. coli JM83 containing pAA130 or pAA152 in the presence or absence of globomycin. Molecular weight standards are noted at the left of FIG. 18 (A and B).
  • FIG. 18A represents lysates of cells containing pAA152 which contains the PBOMP-1 gene. Lanes represent: (1) globomycin absent; and (2) globomycin present.
  • FIG. 18B represents lysates of cells containing pAA130 which contains the PBOMP-2 gene. Lanes represent: (1) globomycin absent; and (2) globomycin present.
  • FIG. 19 graphically illustrates the antibody response obtained when a vaccine formulation comprising PBOMP-1 (5.2 ug) was administered to human adults .
  • FIG. 20 represents reactivity of whole cell lysates of
  • E . coli JM101 or JM103 with monoclonal antibody G-204 Lanes represent: (A) JM103 containing pPX166; (B) JM103 containing pPX160; (C) JM101 containgin pUC19; (D) molecular weight standard displayed in kilodaltons on the left side of the figure; and (E) native PBOMP-1 from H. influenzae.
  • FIG. 21 is a schematic representation of the
  • Plasmid pPX168 was constructed by cleaving the PBOMP-1 coding sequence in pPX167 at the BamHl site in the polylinker and cloning the resulting fragment into the BamHl site of plasmid pINIII- ompA3. Plasmid pPX168 contains a chimeric sequence coding for mature PBOMP-1 linked at the amino termiuns to the signal sequence of E. coli omp A protein.
  • FIG. 22 represents a chromatogram obtained using reverse phase C-4 high performance liquid chromatography of the supernatant fraction of a cytoplasmic extract of E. coli strain PR13 containing plasmid pPX167.
  • FIG. 23A represents SDS-PAGE analysis of signal-less
  • FIG. 23B represents reactivity of the fractions with anti-PBOMP-1 monoclonal antibody. Lanes are as in FIG. 23A.
  • FIG. 24 is a schematic representation of the
  • FIG. 25 represents an SDS-PAGE analysis of whole cell lysates of E. coli JM103 containing pPX163 grown in the presence or absence of IPTG.
  • Lanes represent: (1) molecular weight standards: Kilodaltons; (2) lysate of JM103 containing pPX163 grown without IPTG; and (3) as in Lane 2, grown in the presence of IPTG (5mM) for 4 hours. Arrows show position of three PBOMP-2 reactive bands induced by IPTG.
  • FIG. 26 is a schematic representation of the structure of PBOMP-1 represen'ts hydrophilic regions of the protein and represents reverse turns present in the secondary structure of the protein. The location and size of chemically
  • FIG. 27 represents the ammo acid sequences of the five chemically synthesized PBOMP-1 related peptides.
  • FIG. 28 shows a map of epitopes on the PBOMP-1 protein recognized by monoclonal antibodies to PBOMP-1.
  • FIG. 29 represents reactivity of whole cell lysates of infected E. coli JM103 cells with the capsule deficient H. influenzae strain S2 (lane 4); or of the clinical non-typable H. influenzae strains: 0045E (lane 5), 1939 (lane 6), HST31 (lane 7), and Hib Eagan (lane 8) with anti-PBOMP-2 monoclonal antibody, 61-1.
  • Molecular weight standards (kilodaltons) are shown in lane 2.
  • the reactivities of PBOMP-2 and PBOMP-1 with anti-PBOMP-2 monoclonal antibody 61-1 are shown in lanes
  • FIG. 30 is a schematic representation of the
  • Plasmid pPX183 was
  • FIG. 31 is a schematic representation of the
  • FIG. 32 represents SDS-PAGE analysis of the fatty acylated PBOMP-2: PBOMP-1 fusion protein expressed by E. coli cells containing plasmid pPX199. About 5 ⁇ g of the purified fusion protein was analyzed in a 15% gel (Lane 2).
  • FIG. 33 is a schematic representation of the
  • PBOMP-1 fusion protein coding sequence lacking the PBOMP-2 signal sequence In plasmid pPX512, the PBOMP-2 gene lacking the signal sequence is inserted downstream from the lac
  • Plasmid pPX512 contains a chimeric sequence coding for mature PBOMP-2 sequence, except that the N-terminal cysteine is replaced by methionine, linked at the carboxy terminus to the PBOMP-1 sequence. See text for details of the construction. 5 DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to proteins and peptides related to epitopes of an approximately 16000 dalton molecular weight outer membrane protein of H. influenzae, i.e., PBOMP-1 and of a related approximately 16000 dalton molecular weight outer membrane protein of H. influenzae, i.e., PBOMP-2.
  • the invention is directed further to fusion proteins comprising epitopes of other important proteins of
  • H. influenzae including IgA protease, fimbri and outer membrane proteins.
  • the present invention is also directed to proteins and peptides related to epitopes of a PBOMP-1:
  • PBOMP-2 or a PBOMP-2: PBOMP-1 fusion protein The apparent molecular weights as determined using SDS-PAGE reflect the total molecular weights of the mature (i.e., proteolytically processed) forms, including any post-translational
  • the proteins and peptides of the invention can be produced using recombinant DNA methods or by chemical synthesis.
  • peptides of the invention can be obtained in substantially pure form from cultures of H. influenzae using novel and improved methods of isolation and purification.
  • the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2: PBOMP-1 proteins and peptides specifying epitopes of H. influenzae can be used as immunogens in various vaccine formulations to protect against infection with H. influenzae, an etiological agent of
  • the vaccine formulations are effective against both H. influenzae typable strains including types a, b, c, d, e, and f as well as non-typable H. influenzae strains.
  • the present invention further relates to the nucleotide sequence (s) of the genes encoding the PBOMP-1, PBOMP-2,
  • PBOMP-1 PBOMP-2
  • PBOMP-2 PBOMP-1 proteins as well as the amino acid sequences of the PBOMP-1, PBOMP-2, PBOMP-1:
  • PBOMP-2, and PBOMP-2 PBOMP-1 proteins and polypeptide fragments thereof.
  • recombinant DNA techniques are used to insert nucleotide sequences encoding PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and
  • PBOMP-2 PBOMP-1 epitopes into expression vectors that will direct the expression of these sequences in appropriate host cells. These expression vector host cell systems can be used to produce PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2, and PBOMP-2:
  • PBOMP-1 and PBOMP-2 proteins and peptides may be deduced either (1) from the substantially pure PBOMP-1 protein isolated from H. influenzae as taught herein or (2) from the H. influenzae nucleotide sequences contained in recombinants that express immunogenic PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 related
  • proteins and peptides may then be chemically synthesized and used in synthetic subunit vaccine formulations.
  • PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 or PBOMP-2: PBOMP-1 sequence(s) is a recombinant virus, the virus itself may be used as a vaccine.
  • Infectious recombinant viruses that express the PBOMP-1 and/or PBOMP-2 proteins and peptides and the PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 fusion proteins and peptides, and do not cause disease in a host can be used in live virus vaccine preparations to provide substantial immunity.
  • inactivated virus vaccines can be prepared using "killed" recombinant viruses that express the PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 proteins and peptides.
  • the present invention is further directed to polyvalent antiserum and monoclonal antibody against PBOMP-1, PBOMP-2,
  • PBOMP-1 PBOMP-2
  • PBOMP-2 PBOMP-2
  • PBOMP-2 PBOMP-1 as well as methods for use of such immunoglobulin for passive immunization, and diagnostic assays for H. influenzae.
  • monoclonal antibodies may be generated against PBOMP-1, PBOMP-2, PBOMP-1: PBOMP-2 and/or
  • PBOMP-2 PBOMP-1, related peptides.
  • the method of the invention can be divided into the following stages: (1) isolation and purification of PBOMP-1 protein; (2) partial amino acid sequencing of PBOMP-1; (3) generation of PBOMP-1 and/or PBOMP-2 related peptides which encompass an immunogenic region (s) of PBOMP-1 or PBOMP-2 respectively; (4) molecular cloning of genes or gene fragments encoding PBOMP-1 and PBOMP-2, and PBOMP-1: PBOMP-2 and/or PBOMP-2: PBOMP-1 fusion proteins including insertion of the genes or gene fragments into expression vectors and identification and purification of the recombinant gene products; (5) nucleotide sequencing of the genes encoding PBOMP-1 and PBOMP-2; and (6) determination of the immunopotency of the PBOMP-1, PBOMP-2
  • PBOMP-1 PBOMP-2 and/or PBOMP-2: PBOMP-1 proteins and related products through production of antibodies against purified and recombinant protein and peptide products.
  • the method further encompasses (7) formulation of vaccines and (8) diagnostic assays for detection of PBOMP-1 and PBOMP-2 genes or gene product (and hence H. influenzae) in samples of body fluids.
  • H. influenzae b Eagan In H. influenzae b Eagan and other strains of H.
  • the outer membrane protein PBOMP-1 is associated with the outer membrane-cell wall complex.
  • a necessary step m the purification of PBOMP-1 is the disruption of the bonds which keep the outer membrane proteins in tight association with the outer membrane and cell wall. This can be
  • invention which comprises the following two stages: (1) isolating a PBOMP-1 enriched msoluble cell wall fraction from physically disrupted cells of H. influenzae, and then (2) solubilizing PBOMP-1 from the cell wall fraction by heating in the presence of a detergent which is suitable for administration to a human or digesting the cell wall fraction with lysozyme either in the presence or absence of detergent.
  • novel improved method of the present invention avoids the use of denaturants and reducing agents such as sodium dodecylsulfate and 2-mercaptoethanol (see Munson et al., 1984, Infect. Immun. 49:544-49) which might destroy important epitopes and which are not suitable components for vaccine formulations for administration to humans.
  • denaturants and reducing agents such as sodium dodecylsulfate and 2-mercaptoethanol
  • a total cell membrane fraction may be obtained by differential sedimentation following disruption of H.
  • influenzae cells by methods including but not limited to:
  • the total membrane fraction may be further fractionated into inner and outer membranes by
  • outer membranes are preferably prepared by differential solubilization of inner membranes in 1% (W/V) sarcosyl in 10 mM HEPES-NaOH, pH 7.4.
  • a subfraction enriched in PBOMP-1 can be produced by differential detergent
  • This enrichment can be accomplished, for example, by
  • PBOMP-1 can be solubilized by extraction of the PBOMP-1 enriched fraction with one or any combination of several detergents, including but not limited to
  • PBOMP-1 can be solubilized by disruption of the cell wall in the PBOMP-1 enriched fraction with lysozyme, either in the presence or absence of
  • the detergent is selected from: deoxycholate and polyethoxylate sorbitan monooleate (Tween-80).
  • PBOMP-1 can be isolated by extracting whole H. influenzae cells, outer membrantis or subfractions thereof with one or a combination of detergents including but not limited to: Triton X-100TM, sarcosyl, octylglucoside, nonylglucoside, zwittergent 3-14TM, or zwittergent 3-16TM.
  • This extraction could be performed at 55-60oC or at room temperature in an appropriate buffer system.
  • PBOMP-1 can be achieved by standard methods known in the art including but not limited to: ion exchange, molecular sieve, hydrophobic or reverse phase chromatography, affinity
  • the PBOMP-1 obtained from H. influenzae can be any PBOMP-1 obtained from H. influenzae.
  • fatty acid analysis characterized by fatty acid analysis.
  • such fatty acid analysis revealed the presence of three major fatty acids, i.e., lauric acid, palmitic acid; and a derivative of palmitic acid.
  • the acetylated proteins and peptides of the present invention having a fatty acid moiety covalently attached may be of particular utility for vaccine formulations against H. influenzae.
  • PBOMP-1 and/or PBOMP-2 related peptides which contain an antigenic or immunogenic region(s) of PBOMP-1 or PBOMP-2 respectively may be generated by proteolytic cleavage of the entire PBOMP-1 or PBOMP-2 protein or chemical synthesis of peptide fragments. In the latter method, peptide fragments can be chemically synthesized, for example, using an
  • PBOMP-1 or PBOMP-2 related peptides of the present invention generated either by proteolytic digestion or chemical synthesis may be purified by standard methods known in the art including but not limited to: high pressure liquid chromatography or column chromatography using ion exchange, molecular size, hydrophobic or reverse-phase columns,
  • affinity columns or chromatofocusing, isoelectric focusing or preparative gel electrophoresis affinity columns or chromatofocusing, isoelectric focusing or preparative gel electrophoresis.
  • PBOMP-2 or chemically synthesized PBOMP-1 or PBOMP-2 are determined by techniques known in the art which include but are not limtied to an automated sequenator utilizing the
  • a 16000 dalton molecular weight (MW) OMP has been detected, both by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot analysis in all H. influenzae strains tested (currently several hundred). Monoclonal antibody data indicate that this protein is highly conserved (Murphy et al., 1986, Infect. Immun. 54:774-49). Thus, any H. influenzae strain could serve as the source for the PBOMP genes. Since many H. influenzae strains contain no detectable plasmids or inducible prophages , the PBOMP genes are probably chromosomal .
  • the first step in the molecular cloning of DNA sequences encoding PBOMPs is the isolation of such sequences from H. influenzae chromosomal DNA.
  • DNA encoding H. influenzae genes will be referred to as "Hi DNA”
  • DNA encoding PBOMPs sequences will be referred to as "PBOMP DNA”.
  • Hi DNA DNA encoding H. influenzae genes
  • PBOMP DNA DNA encoding PBOMPs sequences
  • DNAase I to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments may then be separated according to size by standard techniques, including, but not limited to: agarose and
  • Any restriction enzyme or combination of restriction enzymes may be used to generate the Hi DNA fragment(s)
  • the antigenic site of a protein can consist of from about 7 to about 14 ammo acids.
  • a protein of the size of the PBOMP peptides may have many discrete antigenic sites and therefore, many partial PBOMP polypeptide gene sequences could code for an antigenic site. Consequently many restriction enzyme combinations may be used to generate
  • DNA fragments which, when inserted into an appropriate vector are capable of directing the production of PBOMP specific amino acid sequences comprising different antigenic determinants.
  • identification of the specific DNA fragment containing the PBOMP gene may be accomplished in a number of ways.
  • the DNA sequences containing the PBOMP genes may be identified by hybridization of the Hi DNA fragments with a synthetic oligonucleotide probe. Redundant synthetic
  • oligonucleotide probes are constructed based upon the amino acid sequence of peptide fragments of the PBOMP protein.
  • synthetic oligonucleotide probes can be prepared based upon the amino acid sequence of the substantially pure
  • PBOMP-1 protein isolated from H. influenzae as described in Section 5.1 These synthetic probes can be radio-labeled with 32P-adenosme triphosphate and used to screen Hi DNA libraries for clones containing PBOMP-speeific gene sequences
  • the PBOMP gene DNA may be identified and isolated after insertion into a cloning vector in a "shotgun" approach.
  • Vector systems may be either plasmids or modified viruses.
  • Suitable cloning vectors include, but are not limited to the viral vectors such as lambda vector system gtll, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl and other similar systems.
  • the vector system must be compatible with the host cell used.
  • Recombinant molecules can be introduced into cells via transformation, transfeetion or infection.
  • Hi DNA containing a PBOMP gene or gene fragment is inserted into a cloning vector and used to transform
  • the ends of the DNA molecules may be modified. Such modification
  • blunt ends by digesting back single- stranded DNA termini or by filling the single-stranded termini so that the ends can be blunt-end ligated.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini.
  • linkers may comprise specific chemically synthesized oligonucleotides encoding restriction site recognition sequences.
  • DNA modification procedure of Maniatis, (see Maniatis et al., 1982, Molecular
  • sheared DNA is treated with a restriction methylase (for example, M. EcoRI) and ligated to synthetic DNA linkers which encode a restriction site for that enzyme.
  • the DNA is then treated with restriction endonuclease to cleave the terminal linkers (but not the modified internal restriction sites) and ligated to the appropriate vector arms.
  • the cleaved vector and PBOMP DNA fragment may be modified by homopolymeric tailing.
  • the nucleotide sequences coding for PBOMPs or portions thereof are inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • the nucleotide sequences coding for both PBOMP-1 and PBOMP-2 or portions thereof are inserted into an appropriate expression vector as described in the previous sentence, infra.
  • a variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following:
  • bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA bacteriophage DNA, plasmid DNA or cosmid DNA
  • microorganisms such as yeast containing yeast vectors
  • mammalian cell systems infected with virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus.
  • the expression elements of these vectors vary in their strength and
  • any one of a number of suitable transcription and translation elements can be used.
  • RNA polymerase normally binds to the promotor and initiates transcription of a gene or a group of linked genes and regulatory elements (called an operon).
  • Promotors vary in their "strength", i.e., their ability to promote transcription. For the purpose of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence,
  • any one of a number of suitable promotors may be used. For instance, when cloning in E. coli, its
  • RNA promotor trp promotor
  • recA promotor ribosomal RNA
  • promotor the P R and P L promotors of coliphage lambda and others including but not limited to lacUV5, ompF, bla, lpp and the like, may be used to direct high levels of
  • trp-lacUV5 (tac) promotor or other E. coli promotors produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA; for example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D- galactoside).
  • IPTG isopropylthio-beta-D- galactoside
  • trp, pro, etc. are under different controls.
  • the trp operon is induced when tryptophan is absent in the growth media; and the P L promotor of lambda can be induced by an increase in temperature in host cells containing a temperature sensitive lambda represor, e.g., CI857. In this way, greater than 95% of the promotor-directed transcription may be inhibited in uninduced cells.
  • engineered PBOMP protein or peptide thereof may be
  • transformants may be cultured under conditions such that the promotor is not induced, and when the cells reach a suitable density m the growth medium, the promotor can be induced for production of the protein.
  • promotor is constructed by combining the -35 b.p. (-35 region) of the trp promotor and the -10 b.p. (-10 region or
  • enhancer sequences When cloning in a eucaryotic host cell, enhancer sequences (e.g., the 72 bp tandem repeat of SV40 DNA or the retroviral long terminal repeats or LTRs, etc.) may be inserted to increase transcriptional efficiency. Enhancer sequences are a set of eucaryotic DNA elements that appear to increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene.
  • enhancer sequences Unlike the classic promotor elements (e.g., the polymerase binding site and the Goldberg-Hogness "TATA" box) which must be located immediately 5' to the gene, enhancer sequences have a remarkable ability to function upstream from, within, or downstream from eucaryotic genes; therefore, the position of the enhancer sequence with respect to the inserted gene is less critical.
  • classic promotor elements e.g., the polymerase binding site and the Goldberg-Hogness "TATA" box
  • transcription and translation initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in "strength" as measured by the quantity of gene specific messenger RNA and protein synthesized,
  • the DNA expression vector which contains a promotor, may also contain any combination of various components
  • SD Shine-Dalgarno sequence about 7-9 bases 5' to the initiation codon (ATG) to provide a ribosome binding site.
  • any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • insertion of DNA fragments into a vector may be used to ligate a promotor and other control elements into specific sites within the vector.
  • H. influenzae genetic sequences containing those regions coding for the PBOMP proteins or peptides can be .ligated into an expression vector at a specific site in relation to the vector promotor and control elements so that when the recombinant DNA molecule is introduced into a host cell the foreign genetic sequence can be expressed (i.e., transcribed and translated) by the host cell.
  • regions coding for both PBOMP-1 and PBOMP-2 proteins or peptides can be ligated into an expression vector. The relation or orientation of the PBOMP sequences to the vector promoter and control elements will determine whether the genetic sequence expressed is a PBOMP-
  • PBOMP-2 or PBOMP-2 PBOMP-1 fusion protein or peptide fragments, thereof.
  • the recombinant DNA molecule may be introduced into appropriate host cells (including but not limited to bacteria, virus, yeast, mammalian cells or the like) by transformation, transduction or transfection
  • Transformants are selected based upon the expression of one or more appropriate gene markers normally present in the vector, such as ampicillin resistance or tetracycline resistance in pBR322, or thymidine kmase activity m eucaryotic host systems. Expression of such marker genes should indicate that the recombinant DNA molecule is intact and is
  • Expression vectors may be derived from cloning vectors, which usually contain a marker function.
  • cloning vectors may include, but are not limited to the following: SV40 and adenovirus, vaccinia virus vectors, insect viruses such as baculoviruses, yeast vectors,
  • bacteriophage vectors such as lambda gt-WES-lambda B, Charon
  • M13mp7, M13mp8, M13mp9, or plasmid DNA vectors such as pBR322, pAC105, pVA51, pACYC177, pKH47, pACYC184, pUBHO, pMB9, pBR325, Col E1, pSC101, pBR313, pML21, RSF2124, pCR1, RP4, pBR328 and the like.
  • an E. coli plasmid system was chosen as the expression vector.
  • the invention is not limited to the use of such E. coli expression vector.
  • Genetic engineering techniques could also be used to further characterize and/or adapt the cloned gene. For example, site directed mutagenesis of the gene encoding a
  • PBOMP protein could be used to identify regions of the protein responsible for generation of protective antibody responses. It could also be used to modify the protein in regions outside the protective domains, for example, to increase the solubility of the protein to allow easier purification.
  • Expression vectors containing foreign gene inserts can be identified by three general approaches: (1) DNA-DNA hybridization using probes comprising sequences that are homologous to the foreign inserted gene; (2) presence or absence of "marker" gene functions (e.g., resistance to antibiotics, transformation phenotype, thymidine kinase activity, etc.); and (3) expression of inserted sequences based on the physical, immunological or functional properties of the gene product.
  • Marker e.g., resistance to antibiotics, transformation phenotype, thymidine kinase activity, etc.
  • the gene product should be analyzed.
  • Immunological analysis is especially important because the ultimate goal is to use the gene products or recombinant viruses that express such products in vaccine formulations and/or as antigens in diagnostic immunoassays.
  • antisera are available for analyzing immunoreactivity of the product, including, but not limited to polyvalent antisera and monoclonal antibodies described in Section 6.2., infra.
  • the protein or peptide should be immunoreactive whether it results from the expression an entire PBOMP gene sequence, a portion of the gene sequence or from two or more gene sequences which are ligated to direct the production of fusion proteins. This reactivity may be demonstrated by standard immunological techniques, such as radioimmunoprecipitation, radioimmune competition, ELISA or immunoblots.
  • PBOMP related protein produced by a recombinant is
  • the amino acid sequence of the immunoreactive protein can be deduced from the nucleotide sequence of the chimeric gene contained m the recombinant.
  • the protein can be synthesized by standard chemical methods known in the art (e.g., see Hunkapiller et al., 1984, Nature 310: 105-111).
  • such peptides whether produced by recombinant DNA techniques or by chemical synthetic methods, include but are not limited to all or part of the amino acid sequences substantially as depicted in FIG. 11 and/or FIG. 15 including altered
  • residues are substituted for residues withm the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic and glutamic acid.
  • the sequential order of the base pairs can be
  • the actual start and stop signals of the PBOMP genes can be ascertained by analysis of the nucleotide sequence for open reading frames (Rosenberg et al., 1979, Ann. Rev. Genet. 13:319). If more than one open reading frame is found on a particular DNA fragment, the identity of the actual gene could be confirmed by comparing the predicted amino acid sequence of the gene product to the amino acid sequence of the PBOMP. The location of the proper reading frame may also be determined by use of gene fusions.
  • polysaccharide of type b Haemophilus influenzae i.e., PRP shows that the ability of the antibodies to kill the bacteria in in vitro assays and to protect against challenge with Hib in animal model systems is closely correlated with the ability to elicit a protective immune response in human infants.
  • Anti-PBOMP antibodies elicited in response to the PBOMP proteins and peptides of this invention which include but are not limited to PBOMP-1 and/or PBOMP-2 proteins and/or PBOMP- 1: PBOMP-2 and PBOMP-2: PBOMP-1 fusion proteins can be tested using similar in vitro assay systems and animal model system to demonstrate the ability to kill both Hi and Hib cells and to protect in animal model systems from challenge with Hib.
  • antibodies may be elicited in response to chemically synthesized PBOMP peptides or PBOMP peptides generated by proteolytic cleavage of the PBOMP protein.
  • PBOMP peptide fragments would be coupled to a protein carrier, e.g.
  • thyroglobulin bovine serum albumin, diptheria toxin or detoxified toxin (toxoid), tetanus toxin or toxoid,
  • CRM 197 is
  • CRM 197 and native diphtheria toxin are
  • influenzae and non-typable H. influenzae are killed. See Sections 7.1, 9.4, and 10.5 (infra) for illustrative examples of such in vitro bactericidal assays.
  • antibody which is bactericidal against a challenge strain is used to passively immunize infant rats prior to challenge, then they are protected from meningitis and death.
  • Haemophilus, PRP, are protective in the infant rat model system. Passive protection against type b Haemophilus
  • meningitis could be demonstrated by immunizing infant rats with rabbit polyclonal anti-PBOMP antibody and subsequently challenging the rats with a lethal dose of H. influenzae type b. See Section 7.2 (infra) for an illustrative example of such in vivo protective antibody response elicited by the proteins and peptides of the present invention.
  • Anti-PBOMP-1 antibodies are capable of additive protection along with anti-PRP antibodies by use of the infant rat animal model.
  • Anti-PBOMP-1 antibodies diluted to a point at which they no longer are capable of protecting infant rats against
  • Many methods may be used to introduce the vaccine formulations described below into a human or animal. These include, but are not limited to: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous and intranasal routes of administration.
  • One purpose of the present invention is to provide proteins or polypeptide fragments related to outer membrane proteins of H. influenzae, PBOMPs including PBOMP-1, PBOMP-2 and related proteins and peptides as well as PBOMP-1: PBOMP-2 and PBOMP-2: PBOMP-1 fusion proteins and related peptides, which are used as immunogens in a subunit vaccine to protect against meningitis and other disease symptoms of H.
  • Subunit vaccines comprise solely the relevant immunogenic material necessary to immunize a host.
  • Vaccines made from genetically engineered immunogens
  • the PBOMP related protein or fragment thereof may be purified from recombinants that express the PBOMP epitopes.
  • recombinants include any of the previously described bacterial transformants, yeast transformants, or cultured cells infected with recombinant viruses that express the
  • the PBOMP related protein or peptide may be chemically synthesized.
  • the ammo acid sequence of such a protein or peptide can be deduced from the
  • the PBOMP related protein or peptide is isolated in substantially pure form from cultures of H. influenzae
  • Suitable adjuvants include, but are not limited to: surface active substances, e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dicoctadecyl-N'-
  • methoxyhexadecylglycerol, and pluronic polyols plyamines, e.g., pyran, dextransulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine,tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide, aluminum phosphate, etc.
  • the immunogen may also be incorporated into liposomes, or conjugated to
  • polysaccharides and/or other polymers for use in a vaccine formulation.
  • the PBOMP related protein or peptide is a hapten, i.e., a molecule that is antigenic in that it reacts
  • the hapten may be covalently bound to a carrier or immunogenic molecule; for example, a large protein such as protein serum albumin will confer lmmunogenicity to the hapten coupled to it.
  • the hapten-carrier may be formulated for use as a subunit vaccine.
  • Another purpose of the present invention is to provide either a live recombinant viral vaccine or an inactivated recombinant viral vaccine which is used to protect against meningitis and other disease symptoms of H. influenzae.
  • recombinant viruses are prepared that express PBOMP related epitopes (see Sections 5.4. and 5.5., supra). Where the recombinant virus is infectious to the host to be
  • a live vaccine is preferred because multiplication in the host leads to a prolonged stimulus, therefore, conferring substantialy long- iasting immunity.
  • the infectious recombinant virus when introduced into a host can express the PBOMP related protein or polypeptide fragment from its chimeric gene and thereby elicit an immune response against H. influenzae antigens.
  • the live recombinant virus itself may be used in a preventative vaccine against H. influenzae infection. Production of such recombinant virus may involve both in vitro (e. g. , tissue culture cells) and in vivo (e.g., natural host animal) systems.
  • Multivalent live virus vaccines can be prepared from a single or a few infectious recombinant viruses that express epitopes of organisms that cause disease in addition to the epitopes of H. influenzae PBOMPs.
  • a vaccinia virus can be engineered to contain coding sequences for other epitopes in addition to those of H. influenzae PBOMPs.
  • Such a recombinant virus itself can be used as the immunogen in a multivalent vaccine.
  • vaccinia or other viruses each expressing a different gene encoding for differerent epitopes of PBOMPs and/or other epitopes of other disease causing organisms can be formulated in a multivalent vaccine.
  • an inactivated virus vaccine formulation may be prepared.
  • Inactivated vaccines are "dead" in the sense that their infectivity has been destroyed, usually by chemical treatment (e.g., formaldehyde). Ideally, the infectivity of the virus is destroyed without affecting the proteins which carry the immunogenicity of the virus.
  • chemical treatment e.g., formaldehyde
  • large quantities of the recombinant virus expressing the PBOMP related protein or polypeptide must be grown in culture to provide the necessary quantity of relevant antigens.
  • a mixture of inactivated viruses which express different epitopes may be used for the formulation of "multivalent” vaccines. In certain instances, these "multivalent" inactivated vaccines may be preferable to live vaccine formulation because of potential difficulties with mutual interference of live viruses administered
  • the inactivated recombinant virus or mixture of viruses should be formulated in a suitable adjuvant in order to enhance the immunological response to the antigens.
  • suitable adjuvants include, but are not limited to: surface active substances, e.g., hexadecylamine, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N, N-dicoctadecyl-N'- N-bis (2-hydroxyethyl-propane diamine),
  • methoxyhexadecylglycerol, and pluronic polyols plyamines, e.g., pyran, dextransulfate, poly IC, polyacrylic acid, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g., aluminum hydroxide, aluminum phosphate, etc.
  • the vaccine formulations can be used to produce antibodies for use in passive
  • Human immunoglobulin is preferred in human medicine because a heterologous immunoglobulin may provoke an immune response to its foreign immunogenic components.
  • passive immunization could be used on an emergency basis for immediate protection of unimmunized individuals exposed to special risks, e.g., young children exposed to contact with bacterial meningitis patients. Alternatively, these
  • antibodies can be used m the production of anti-idiotypic antibody, which in turn can be used as an antigen to
  • Yet another purpose of the present invention is to provide reagents for use in diagnostic assays for the
  • the PBOMP related proteins and peptides of the present invention may be used as antigens in immunoassays for the detection of H. influenzae in various patient tissues and body fluids including, but not limited to: blood, spinal fluid, sputum, etc.
  • the antigens of the present invention may be used in any immunoassay system known in the art including, but not limited to: radioimmunoassays, ELISA assays, "sandwich” assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays and
  • immunoelectrophoresis assays to name but a few.
  • nucleotide sequence of the genes encoding the PBOMP related protein and peptides of the present invention may be used as probes in nucleic acid hybridization assays for the detection of H. influenzae in various patient body fluids, including but not limited to: blood, spinal fluid, sputum, etc.
  • nucleotide sequences of the present invention may be used in any nucleic acid hybridization assay system known in the art including, but not limited to: Southern blots
  • influenzae as follows:
  • H. influenzae Eagan was grown overnight on either brain heart infusion medium containing 10 ug/ml hemin and 1 ug/ml
  • the cell pellet was weighed and suspended in 10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA, with a volume of buffer equal to about five times the wet weight of the cells.
  • the cell suspension was then sonicated for 5 minutes in an ice bath in 100 ml aliquots with a Branson Model 350 sonifier cell disruptor (Branson Sonic Power, Danbury, CT) at 60% power on a pulse setting. Following sonication, the disrupted cell suspension was centrifuged at 10,000 x g for 5 minutes at 4oC to remove unbroken cells. The sedimented unbroken cells were then weighed and re-sonicated as before in a volume of 10 mM
  • the total membrane fraction was obtained as a pellet following addition of sufficient NaCl to provide a final concentration of 0.5 M NaCl and ultracentrifugation of the broken cellular material at 100,000 x g for about 1 hour.
  • An outer membrane-cell wall complex was then obtained by removing the inner membrane components from the total membrane fraction by repeated extraction of the total membrane fraction with 1% sarcosyl, in 10 mM HEPES-NaOH, pH 7.4.
  • the insoluble residue containing the outer membrane cell wall fraction was isolated by centrifugation at 350,000 x g for 30 minutes, suspended in 50 mM Tris pH 8.0, 5 mM
  • octylglucoside and sarcosyl is a PBOMP-1-cell wall complex.
  • PBOMP-1 was solubilized by two methods: (1) heating to
  • dodecylmaltoside, zwittergent 3-14TM and zwittergent 3-16TM) are effective in the heat dependent solubilzation as well as the lysozyme induced solubilization. Additionally,
  • octylglucoside is very effective in the lysozyme induced solubilizations and was used routinely at 1% (w/v) final concentration. From 40 g wet weight cells, it was possible typically to isolate about 8 mg of PBOMP-1, substantially free from other cell wall components . This substantially pure PBOMP-1 preparation was analyzed in an SDS PAGE system to determine the relative molecular weight of the reduced denatured form of this protein and to assess its purity
  • the stacking gel contained 4.8% acrylamide with the same ratio of acrylamide to bis, 125 mM Tris, HCl (pH 7.0), and 0.1% SDS per gel.
  • PBOMP-1 Further purification of PBOMP-1 can be achieved by standard methods such as ion exchange chromatography,
  • a proteolytic digest of the 16,000 dalton PBOMP-1 obtained using trypsin, at 27°C for 1 hour was separated by reverse phase high pressure liquid chromatography (RP-HPLC) using a C18 column.
  • RP-HPLC reverse phase high pressure liquid chromatography
  • a large hydrophobic peptide peak (T9) was isolated and subsequently immobilized on a polybrene- coated glass fiber paper prior to the start of amino acid sequencing.
  • the T9 peptide was sequenced by Edman degradation
  • the PTHs were eluted at room temperature with a sodium acetate-acetonitrile gradient and detected at 270 nanometers with a variable UV wavelength detector.
  • the T9 peptide is very hydrophobic containing 8
  • PBOMP-1 is unusual in that it contains 13 tyrosines, but no methionine or tryptophan. 6.1.2 CHARACTERIZATION OF PBOMP-1
  • PBOMP-1 protein isolated as described above in Section 6.1. was extracted exhaustively with a mixture of organic solvents, i.e., chloroform:methanol (2:1) and with deoxycholate detergent to remove any trace contaminants of endogenous lipids , phospholipids , etc.
  • the denuded protein was obtained either by acetone precipitation or by exhaustive dialysis and dried by lyophilization.
  • a known amount of nonadecanoic acid was added as an internal standard to the dried purified PBOMP-1 (1-3 mg) and the mixture was hydrolyzed with 200 ul of 4 N HCl at 110°C for 4 hours under a nitrogen atmosphere. Such acid hydrolysis released amide- or ester- linked fatty acids.
  • hyrdolysate diluted to 2 ml with water, was extracted three times with an equal volume of hexane.
  • the combined hexane phase was washed twice with an equal volume of saline and then dried over sodium sulfate.
  • the fatty acids were
  • C16' is perhaps a branched chain fatty acid having 16 carbon atoms.
  • PBOMP-1 phosphate buffered saline
  • PBOMP-1 were minced by passing them through a 25 gauge needle in PBS. The fragments were injected intramuscularly into New Zealand white rabbits at multiple sites. Each rabbit
  • Rabbits were injected intramuscularly with approximately 20 ug of PBOMP-1 in Freund's adjuvant. Animals were reimmunized two weeks and three weeks following the initial immunization and bled one week following the last immunization.
  • Substantially pure PBOMP-2 was used as an immunogen to prepare anti-PBOMP-2 antibodies.
  • PBOMP-2 bands were excised from the gels and dialyzed against phosphate buffered saline PBS until equilibrated.
  • the acrylamide gel fragments containing PBOMP-2 were minced by passing them through a 25 gauge needle in PBS.
  • the minced PBOMP-2 gel fragments were mixed with complete Freund's adjuvant and emusified. Rabbits were injected intramuscularly with approximately 10 ug of PBOMP-2 in Freund's adjuvant. Animals were boosted with 10 ug of the same preparation in incomplete Freund's adjuvant four weeks after the primary inoculation.
  • Hybridoma cell lines secreting antibodies to PBOMP-1 or PBOMP-2 were obtained by fusion of mouse myeloma cell line X63.Ag8.6543 with spleen cells obtained from a C57/B1 mouse immunized against H. influenzae as follows: A female C57/B1 mouse was injected intraperitioneally four times over a period of two months with 1 x 10 6 H. influenzae strain S2 cells. Three months later, the mouse was immunized with substantially pure PBOMP-1 or PBOMP-2 isolated from an SDS- PAGE band as described in Section 6.2.1. One month later, the mouse received an intravenous injection of total outer membranes from S2. Cell fusion was performed on the fourth day post- intravenous injection by standard procedures common to those of skill in the field (for example, Gefter et al., 1977, Somat. Cell. Genet 3:231-36).
  • Hybridoma cell culture supernatants were screened by a standard ELISA using H. influenzae outer membrane proteins as antigens. Assays were performed in 96 well polystyrene plates coated overnight at 4°C with OMPs.
  • hybridomas, designated 61-1 was found to be specific for
  • influenzae PBOMP-1 may also protect against some E. coli infections.
  • the anti-PBOMP-2 monoclonal antibody, 61-1 was found to cross-react weakly with a protein of about 15000 daltons from E. coli, but showed no detectable cross-reactivity with PBOMP-1.
  • Restriction enzyme digestions were carried out by suspending DNA in the appropriate restriction buffer, adding restriction endonuclease, and incubating for an appropriate period of time to ensure complete digestion.
  • One unit of enzyme is defined as the amount required to completely digest 1.0 ug of phage lambda DNA in 1 hour in a total reaction mixture of 20 ul volume. Buffers used with the various enzymes are listed below: Low salt buffer used for Clal, Hpal, Hpall, and Kpnl digestions consisted of: 10 mM Tris (pH 8.0), 10 mM MgCl 2 and 10 mM dithiothreitol (DTT).
  • Medium salt buffer used for Alul, Aval, EcoRII, EcoRV, Haell, Haelll, HincIII, Hindlll, Pstl, Sau3AI, Sphl, Sstl, Sstll, Taql, and Xhol digestions consisted of: 50 mM Tris (pH 8.0), 10 mM MgCl 2 , 50 mM NaCl, and 10 mM DTT.
  • High salt buffer used for BamHl, EcoRI, Pvul, Sall and Xbal digestions consisted of: 50 mM Tris (pH 8.0), 10 mM
  • the buffer used for Smal digestions consisted of: 10 mM
  • Tris (pH 8.0), 20 mM KCl, 10 mM MgCl 2 , and 10 mM DTT.
  • the buffer used for Seal digestions was: 100mM NaCl: 10 mM Tris HCl (pH 7.4); 10 mM MgCl 2 and 1 mM DTT. All restriction digestions were carried out at 37oC except Taql, which was carried out at 60oC.
  • T4 DNA ligase was purchased from BRL (Bethesda, MD), United States Biochemicals (Cleveland, OH) or Boehringer
  • T4 DNA ligase is defined as the amount required to yield 50% ligation of Hindlll fragments of bacteriophage lambda DNA in 30 minutes at 16oC m 20 ul volume ligase buffer at a 5'-DNA termini
  • DNA ligations were performed in ligase buffer consisting of: 50 mM Tris (pH
  • Proteins were fixed to nitrocellulose sheets for immuno blot analysis by various techniques, depending on the
  • Phage plaques were transferred from agar plates by gently placing a sterile 8.1 cm diameter nitrocellulose disc onto the surface of a 10 cm diameter phage titer plate. The sheet was allowed to wet completely, positions were marked by punching through the filter with a sterile needle, and the filter was lifted after two minutes.
  • Colony blots were performed by transferring bacterial colonies to a nitrocellulose sheet, allowing the colonies to grow by placing the sheet (colony side up) on nutrient agar for 4 to 6 hours, and exposing the sheet to chloroform vapor for 30 minutes to lyse the colonies.
  • Protein gel transfers were performed by placing an SDS-PAGE gel containing the protein mixture to be analyzed on a nitrocellulose sheet and applying horizontal electrophoresis in a Hoeffer Transphor apparatus at 0.5 A for 14 hours in 25 mM Tris 0.38M glycine pH 8.8 buffer.
  • TnPhoA transposition of TnPhoA such that the PhoA gene is fused in frame into an actively transcribed gene containing a membrane transport signal peptide.
  • Such transpositions were detected by plating cells in the presence of 40 ug/ml 5-Bromo-4-Chloro-3-Indolyl Phosphate (XP, Sigma
  • DNA filter hybridization analysis was carried out according to the procedure of Southern (1975, J. Mol Biol. 98: 508). DNA to be analyzed by filter hybridization was digested with appropriate restriction endonuclease (s) and separated by agarose gel electrophoresis in 0.8% Agarose
  • DNA in the gel was denatured by soaking the gel in 1.5 M NaCl/0.5 M NaOH for 1 hour and neutralized by soaking in 1.5 M NaCl/1.0 M Tris-HCl for 1 hour. Denatured DNA was transferred to nitrocellulose filter paper by blotting. Following transfer of DNA, filter were washed with 6 X SSC (prepared by dilution from a 20X SSC stock containing 175.5 g NaCl and 88.2 g Na citrate/liter) and air dried. DNA fragments were fixed to the filter by baking at 80°C for 2 hours under vacuum.
  • 6 X SSC prepared by dilution from a 20X SSC stock containing 175.5 g NaCl and 88.2 g Na citrate/liter
  • DNA hybridization probes were prepared by nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick nick
  • DNA for the probe (1-2 ug) was dissolved in 100 ul nick-translation buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO 4 , 10 mM DTT, 5 ug/ml bovine serum albumin, and 20 urn each dGTP, dCTP, and dTTP).
  • 100 uCi of alpha 32 P-dATP (Amersham, 2-3000
  • E. coli DNA polymerase I (Boehringer) were added and the mixture incubated at 15°C for 45 minutes. The reaction was stopped by the addition of EDTA to 50 mM and heating to 65oC for 10 minutes to inactivate the enzymes.
  • the labeled DNA was precipitated by addition of three volumes of ethanol and resuspended to 50 ul of 0.3 M ammonium acetate
  • filters with bound DNA were wetted with 6 x SSC and prehybridized with 6 X SSC/ 0.5% SDS/5X
  • Denhardt's solution/100 ug/ml tRNA at 68o C for 2 hours to block excess binding capacity of the filter (1 X Denhardt's solution is .02% Ficoll, 0.02% polyvinylpyrrolidone, .02% bovine serum albumin in water).
  • the hybridization reaction was carried out in the same buffer to which 0.01 M EDTA and
  • H. influenzae chromosomal DNA for cloning of the PBOMP genes was either H. influenzae KW20b (HiKW20b), a derivative of a non-encapsulated Rd stain of Hi transformed to type b+ by DNA from strain b-Eagan (Moxon et al, 1984, Clin. Invest. 73 : 298-306) or H . influenzae S2, (Hi S2), a spontaneous capsule-minus mutant of Hib Eagan.
  • chromosomal DNA from Hi was sheared to an average length of about 15000 base pairs (bp), blunt ended by treatment with T4 DNA polymerase, modified with EcoRI DNA methylase, ligated to synthetic EcoRI linkers, and cloned into the recombinant Lambda phage vector
  • This plasmid is a derivative of pLG339
  • E. coli containing recombinant plasmids were screened for production of PBOMPs using a pooled mixture of monoclonal antibodies or polyclonal anti-PBOMP-1 antiserum.
  • Chromosomal DNA from a Hi S2 was partially digested with restriction endonuclease Sau3A (BRL, Bethesda, MD).
  • Plasmid pGD103 DNA was digested with BamHl endonuclease and treated with calf alkaline phosphatase (Boehringer, Indianapolis, IN) (1 Unit/ug DNA, 37°C x 30 minutes in restriction buffer). DNA was purified from the reaction mixture by phenol extraction and ethanol precipitation and resuspended in TE buffer. Since BamHl and
  • Sau3A restriction enzymes form cohesive ends, no further treatment of DNAs prior to ligation was necessary.
  • colonies were picked, amplified individually, and stored frozen at -70°C in LB broth containing 18% sterile glycerol in 96-well microtiter dishes.
  • High molecular weight chromosomal DNA from Hi KW20b was suspended in TE buffer at a concentration of 200 ug/ml and sheared to an average length of 15000 bp by passage through a
  • DNA was then modified with EcoRI DNA methylase (1 U/ug DNA) (BRL Bethesda, MD), in 100 mM Tris (pH 8.0), 10 mM EDTA, 0.1 mM S-adenosyl-methionine) for 3 hours at 37°C. Methylation of DNA was verified by removing 1 ug of
  • modified Hi DNA was ligated to 1 ug chemically synthesized EcoRI linkers (BRL Bethesda, MD) in a 100 ul reaction mixture using T4 DNA ligase (5U). After 18 hours, the reaction was stopped by heating to 60oC for 20 minutes, NaCl was added to a final concentration of 150 mM, and the mixture was digested with 10 U EcoRI for 6 hours. Modified Hi DNA plus linker was separated from cleaved and unligated linkers by agarose gel electrophoresis as describe above.
  • Phage were removed from the interface and dialyzed against TMG. Titering of the phage thus prepared indicated a library of 25-30,000 independent clones of the Hi genome had been generated.
  • the phage library was amplified by plate amplification using E. coli KH802 as a phage host to yield 5 ml of phage suspension containing 10 -9 plaque forming units
  • the Hi plasmid library was transferred to
  • FIG. 4 shows a restriction map of pAA152 which contains a 4.2 Kb Hi DNA insert in vector pGD103.
  • the amplified phage library prepared as described in section 6.5.1. was diluted to 1-2000 PFU in one ml TMG and 50 ul of E. coli KH802 (5 x 10 9 cells/ml) were added. The mixture was incubated at 37oC for 20 minutes and plated with
  • NZ Amine A 5.0 g NaCl, 2.0 g MgSO4.7H 2 O, 5 g Yeast Extract,
  • phage One positive phage, designated lambda 16-3 was selected for further analysis. This phage isolate was amplified by growth m E. coli KH802 in NZYCM broth, recovered by
  • the lambda 16-3 DNA was digested with EcoRI and a partial physical map of the Hi chromosomal insert was
  • FIG. 7 represents a restriction map of this plasmid having an 5.7 Kb fragment from Hi DNA cloned into pGD103 plasmid.
  • the labelled 16000 dalton protein was specifically
  • Plasmid pAA130 directs the expression of a 16000 dalton molecular weight PBOMP.
  • the 781 base pair BstEII-XmnI fragment was cloned by isolating the fragment from a low melting point agarose gel, filling in the BstEII end with Klenow fragment of DNA
  • the large EcoRI-PvuII fragment of pAA130 was ligated with the EcoRI-PvuII fragment of pLG339 to generate a new tetracycline resistant plasmid designated pAA136.
  • This plasmid expressed the PBOMP as verified by Western blots.
  • This plasmid was transformed into an E. coli strain with deletion of the chromosomal alkaline phosphatase gene (phoA) and carrying the transposible element TnPhoA.
  • phoA chromosomal alkaline phosphatase gene
  • All three TnPho insertions were in the same orientation indicating that transcription of the PBOMP gene is directed from the BstEII site towards the Xmnl site in pAA136.
  • All three TnPhoA transpositions resulted in loss of the 16000 protein detected by polyclonal anti-PBOMP-1 antiserum as detected by Western blots.
  • One fusion generated a new band on Western blots at 60000 dalton which was detected by polyclonal anti-PBOMP-1 antiserum. This size is within the predicted range of fusion proteins that might be generated by fusion of alkaline phosphatase (45000 daltons MW) to a 16000 dalton MW protein.
  • Restoration of PhoA activity in these transpositions verifies that the PBOMP protein contains a peptide signal for membrane transport; and hence, is probably a membrane protein.
  • TnPho fusions were sequenced by subcloning the junction between TnPhoA and the Hi cloned DNA sequences into
  • the nucleotide sequence of the PBOMP gene expressed by pAA152 was obtained by dideoxynucleotide sequencing (Sanger et al., 1978, Proc. Nat'l Acad. Sci USA 74:5463-5467) of the
  • the 737 Bglll-BamHI fragment of pAA152 contains a single open reading frame (ORF) coding for a polypeptide of 153 amino acids (FIG. 11).
  • ORF open reading frame
  • the amino acid composition of the PBOMP gene determined from the DNA sequence closely matches the amino acid composition of the PBOMP-1 purified protein (see Tables 1 and 2).
  • the PBOMP-1 gene has an internal peptide
  • the nucleotide sequence of the PBOMP gene of pAA130 was determined by dideoxynucleotide sequencing (Sanger, et al., supra) of the 789 base pair BstEII-XmnI fragment of pAA130 after subcloning into M13 mpl8 and mpl9 phage.
  • recombinant phage are designated M18001 and M19001
  • sequencing primer New England Biolabs was used to determine the sequence from both ends of the BstEII-Xmnl fragment (see FIG. 13).
  • Two additional oligonucleotides were synthesized and used as primers for dideoxynucleotide sequencing (M18PRI, M19PR2). All other sequencing primers were made at Kir Biologies, Rochester, N.Y. on an Applied Biosystems 380 B DNA synthesizer. The primers were made on a 1 umole controlled pore glass column with beta-cyanoethyl phosphate protecting group chemistry. The yield of oligonucleotide was
  • sequence data from the TnPhoA fusions in pAA130 demonstrated that all three transpositions were into the reading frame of the 154 amino acid polypeptide.
  • amino acid composition of the proposed mature gene product as deduced from the DNA sequence of the ORF of pAA130 differs significantly from that determined by amino acid analysis of purified PBOMP-1 (Tables 1 and 2).
  • PBOMP-1 isolated from H. influenzae has covalently attached fatty acids, including lauric acid, palmitic acid and a derivative of palmitic acid, and hence can be classified as a bacterial lipoprotein.
  • the following experiments were performed to investigate whether PBOMPs expressed by recombinant E. coli also exist as lipoproteins. Two different in vivo methods were used to verify the lipoprotein nature of the expressed
  • recombinant plasmids expressing PBOMPs were cultured in the presence of a radioactively labelled fatty acid. Under such conditions, any lipoprotein formed containing the covalently attached fatty acid will be specifically radiolabeled.
  • E. coli JM83 cells containing either plasmid pAA152 expressing PBOMP-1 or plasmid pAA130 expressing PBOMP-2 were grown for 2 hours in M9-minimal medium containing 50 uCi/ml
  • cells containing recombinant plasmids expressing PBOMPs were cultured in the presence of globomycin, an antibiotic known to specifically block processing of all known bacterial liproproteins by inhibition of the liproprotein signal peptidase (Inukai et al., 1978, J. Antibiotics (Tokyo) 31:1203-1205).
  • E. coli JM83 cells containing either pAA152 or pAA130 were cultured in the presence of 25 ug/ml globomycin
  • Anti-PBOMP-1 polyclonal rabbit antisera prepared as described in Section 6.2., were examined for their ability to kill Hib and Hi in an in vitro complement mediated
  • PCCS pre-collostral calf serum
  • NRS normal rabbit serum
  • phosphate- buffered saline 20 mM phosphate buffer (pH 7.4), 0.15 M NaCl containing 0.15 mM MgCl 2 and 0.5 mM CaCl 2 (PCM)].
  • Bacterial strains to be tested were grown in BHI-XV until they reached a concentration of 1 x 10 9 cells/ml as measured by optical density at 490 mm. Bacteria were diluted to a final
  • microtiter plate (Costar). The microtiter plate was removed from ice and 20 ul of test diluted bacteria were added to each well. Wells containing no antibody served as negative controls. After 30 minutes incubation at 37oC, 800 ul of BHI-XV, containing 0.75% agar at 56oC, were added to each well and allowed to solidify at room temperature. The plates were incubated overnight at 37oC and read the next day.
  • the BC titer of an antisera was read as the reciprocal of the highest dilution capable of killing 50% of the test bacteria as compared to non-antibody control wells.
  • the anti-PBOMP-1 was tested for bactericidal (BC) activity against several Hib clinical and laboratory isolates and the results shown in Table 3.
  • anti-PBOMP-1 antibody had BC activity against a wide variety of clinical isolates both typable (e.g. a, b, c) and non-typable H. influenzae strains.
  • typable e.g. a, b, c
  • non-typable H. influenzae strains One hundred and twelve out of 112 Hib clinical isolates were killed by anti-PBOMP-1 antisera. These strains were isolated in the Southeastern U.S., the Northeastern U.S. and Western Canada.
  • the LPS lowered the titer of the anti-PRP two-fold, the titer of the anti-PBOMP-1 against Hi four-fold and the titer of anti-PBOMP-1 against Hib not at all. While the LPS reduced the BC activity of anti-PBOMP-1, it did not eliminate it. Some of the observed reduction was undoubtably the result of anti-complementary activity of the LPS, as demonstrated by the reduction of the anti-PRP BC titer.
  • Hib PRP polysaccharide (Farr-type RIA) (see, Farr, 1958, J. Infect. Dis. 103:239-262 for description of Farr-type RIA).
  • Results obtained in the PBOMP-1 ELISA assay are illustrated in FIG. 19.
  • rPBOMP-1 A recombinant signal-less PBOMP-1 (herein designated rPBOMP-1) obtained from E. coli PR13 cells containing plasmid pPX167 as described infra in Section 8.1 was used as an immunogen to immunize white New Zealand rabbits.
  • rPBOMP-1 was either emulsified in incomplete Freund's
  • rPBOMP-1 was bound to alum by mixing rPBOMP-1 at a concentration of 500 ug/ml in 0.85% saline with alum at a concentration of 500 ug/ml at a 1:1 ratio. The mixture was shaken at 37°C for 3 hours and the alum pelleted by centrifugation. Supernatent was assayed by BCA protein assay (Pierce Chem. Co., Chicago, IL) for unbound protein. None was detected. Alum was resuspended in physiological saline at 500 ug/ml. rPBOMP-1 was emulsified in IFA (Difco) in a 1:1 ratio. Animals were immunized intramuscularly with 50 ug of either preparation at 4 week intervals. Animals were bled at weeks 0, 6, and before each immunization.
  • the anti-rPBOMP-1 polyclonal rabbit antisera obtained were examined for the ability to kill Hi in an in vitro
  • test bacterium was nontypable H.
  • influenzae strain S-2 Results are shown in Table 8.
  • the BC titer of an antisera was read as the reciprocal of the highest dilution capable of killing 50% of the test bacteria as compared to non-antibody control wells.
  • the rabbit immunized with rPBOMP-1 in IFA had a titer of 1:160 and the rabbit immunized with rPBOMP-1 on alum had a titer of 1:80.
  • Hyperimmune antiserum was obtained after the rabbit received multiple doses of native PBOMP-1.
  • the hyperimmune rabbit anti-PBOMP-1 serum had a titer of 1:160.
  • the PBOMP-1 protein is expressed from the 737 bp BamHI-Bglll fragment of pAA152, presumably under control of its native promoter. The sequence contains a good consensus ribosome binding site and initiation codon of the PBOMP-1 gene. While PBOMP-1 expressed in E. coli with plasmids containing this fragment was easily detected by Western blot analysis, the amount of such protein produced was less than 1% of cell protein, i.e., less than the amount of PBOMP-1 made in H. influenzae cells containing the native gene.
  • the cloned gene was placed under the control of promoters lac and lambda P L known to yield high protein production. Promoters were linked to the BstNI site upstream of the PBOMP-1 initiation codon (FIG. 4A). Cleavage at this site removes the native PBOMP-1 promoter but leaves the ribosome binding site intact.
  • the 739 bp BamHI-Bglll fragment of pAA152 carrying the PBOMP-1 gene was cloned into the BamHl site of lac promoter of plasmid pUC19.
  • One clone carrying the PBOMP-1 gene in the same orientation as the lac promoter was designated pPX160.
  • Expression of PBOMP-1 from pPX160 in E. coli JM103 was under regulation of the native promoter not under lac regulation; apparently due to a transcription termination signal in the 240 bp between the Bglll site and the translation initiation codon of PBOMP-1. Plasmid pPX160 was then cleaved with
  • the resulting plasmid designated pPX166 w0as found by Western blot to express PBOMP-1 under regulation of the lac promoter in E. coli JM103.
  • Plasmids pPX160 and pPX166 were transformed into several E. coli strains containing mutations reported to stabilize foreign proteins. These include ATP-dependent protease (Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response (htp), and an mRNA-stabilizing mutation (pnp). In addition, since Ion-) mutations, heat shock response
  • a modified PBOMP-1 gene was created by removing the native signal sequence of the gene.
  • Such a construction offers two potential advantages over native PBOMP-1 protein.
  • the signal-less PBOMP-1 may not be transported to the membrane, and hence, toxicity effects due to overexpression of PBOMP-1 may be lessened.
  • signal-less PBOMP-1 may not require use of detergents for isolation or storage in solution.
  • FIG. 21 Construction of a signal-less form of PBOMP-1 is illustrated in FIG. 21.
  • the PBOMP-1 gene from plasmid pPX160 was cleaved at codon 19 with Alul restriction endonuclease. The resulting fragment was ligated to the Smal restriction site within the pUC19 polylinker region. The resulting gene expressed a hybrid protein containing all of the amino acids sequence of native PBOMP-1 plus an additional 18 amino acids from the pUC19 polylinker region at the amino terminus.
  • This plasmid was designated pPX167.
  • plasmid pPX167 contains a hybrid gene which encodes mature PBOMP-1 linked at the amino terminus to the signal sequence of E. coli OMP A protein. This hybrid product is processed through the membrane via the OMP A signal sequence to generate a mature PBOMP-1 lacking lipoprotein modification and containing eight additional amino acids at its amino terminus.
  • Plasmids pPX167 and pPX168 were transformed into E. coli JM103 and tested for recombinant PBOMP-1 synthesis. By SDS-PAGE Western blot analysis, both plasmids were shown to encode proteins which were recognized by polyclonal and monoclonal anti-PBOMP-1 antisera.
  • the modified PBOMP-1 synthesized from pPX167 was inducible with isopropylthio- beta-d-galactopyranoside (IPTG), was located in the cell cytoplasm, and was soluble in the absence of detergents. The modified PBOMP-1 was not detectable by Coomassie blue
  • Plasmids pPX167 and pPX168 were also tested in a variety of E. coli strains for levels of expression. The most successful combination tested was the pPX167 chimeric plasmid transformed into E. coli PR13, a strain containing the mRNA stabilizing mutation pnp. In this strain,
  • recombinant PBOMP-1 is expressed under control of the lac promoter at about 2-3% of total cell protein after lac
  • This recombinant PBOMP-1 is expressed as a cytoplasmic fusion protein containing about 17 amino acids from the lac alpha-peptide and multiple cloning sequence fused to the amino terminus of the PBOMP-1 gene. 8.1.1 PURIFICATION AND CHARACTERIZA- TION OF SIGNAL-LESS PBOMP-1
  • Unbroken cells were removed by centrifugation at 10,000 rpm in an SS-34 rotor at 4°C for 10 minutes. Total cell membranes were removed by centrifugation at 55,000 rpm in a 60Ti rotor for 30 minutes at 4°C.
  • SDS-PAGE was performed as described above herein, on the cytoplasmic extract, the DEAE eluate and the reverse phase eluate obtained from the E. coli PR13 cells containing plasmid pPX167. After electrophoresis, the gels were stained with Coomassie brilliant blue stain for about 2 hours.
  • rPBOMP-1 is the major protein present in the cytoplasmic extract of cells of E. coli PR13 containing plasmid pPX167 when stained with Coomassie stain
  • FIG. 23A As estimated by Western blot reactivity, greater than 95% of the rPBOMP-1 is located in the
  • the results indicate that the rPBOMP-1 obtained from those cells is soluble in 10 mM Tris-Hcl, pH 7.5 without detergent. This represents a departure from the PBOMP-1 obtained from Hib cells as a xipoprotein.
  • rPBOMP-1 obtained from E. coli PR13 cells containing pPX167 may exist in aqueous solutions as a complex either with itself or with other cytoplasmic proteins.
  • gel filtration chromatography using acrylamide-agarose polymers or Sephadex beads was performed using this rPBOMP-1, the rPBOMP-1 eluted at an apparent molecular mass of greater than 100,000 daltons (data not shown).
  • rPBOMP-1 did not elute as a single peak at a particular salt concentration.
  • the rPBOMP-1 eluted over a range of NaCl concentrations from approximately 20 mM to 75 mM in 10 mM Tris, pH 7.5. While the eluted rPBOMP-1 was one of many eluted proteins in the 80 mM NaCl wash, a significant number of cytoplasmic proteins remained bound to the column at salt concentrations greater than 80 mM.
  • DEAE chromatography as described above was used as a
  • the amino acid sequence of rPBOMP-1 indicates that it sould be a relatively hydrophobic protein. While it was soluble in the cytoplasmic extract without detergents, it was expected that the rPBOMP-1 would be more hydrophobic than most of the cytoplasmic proteins.
  • the DEAE eluate containing the rPBOMP-1 was thus chromatographed on a C-4 hydrophobic0i.nteraction column as described above and eluted using a gradient of increasing acetonitrile. In this system, more hydrophobic proteins should be bound more tightly to the column and thus elute in higher concentrations of
  • lane 3 the rPBOMP-1 peak was pure as determined by Coomassie brilliant blue R-250 staining of 10 ug of protein after SDS-PAGE.
  • the peak showed two bands, a major and a minor band, on Coomassie staining, both of which reacted with monoclonal antibody to PBOMP-1 which recognizes the amino terminal 20 amino acids of PBOMP-1 (FIG.
  • purified rPBOMP-1 was soluble in aqueous solvents without detergent.
  • rPBOMP-1 obtained from E. coli PR13 cells containing pPX167 either emulsified in incomplete Freunds adjuvant or bound to aluminum hydroxide.
  • rPBOMP-1 was bound to alum by mixing rPBOMP-1 at a concentration of 500 ug/ml in
  • saline 0.85% saline with alum at a concentration of 500 ug/ml at a 1:1 ratio.
  • the mixture was shaken at 37°C for 3 hours and the alum pelleted by centrifugation.
  • Supernatent was assayed by BCA protein assay (Pierce Chem. Co, Chicago, IL) for unbound protein. None was detected.
  • Alum was resuspended in physiological saline at 500 ug/ml.
  • rPBOMP-1 was emulsified0i.n i.ncomplete Freund's adjuvant (Difco) in a 1:1 ratio.
  • PBOMP-2 is also expressed at low levels (less than 1% of cell protein) from plasmid pAA130. Expression of PBOMP-2 is apparently under control of its native promoter.
  • the PBOMP-2 5jene in pAA130 was cleaved at a unique Sspl restriction site 5 bases 5' to the PBOMP-2 initiation codon and ligated to the Smal site of pUC19 (FIG. 24).
  • the ligation results in a hybrid open reading frame (ORF) containing the entire PBOMP-2 ORF (including the signal sequence) plus 18 codons from pUC19 Qmd 2 codons from the gene fusion site.
  • ORF open reading frame
  • the protein product of this fusion gene has a predicted molecular weight of
  • the 17,500 dalton apparent MW band is the predicted PUC19/PBOMP-2 hybrid protein
  • the upper 15,000 dalton MW band starts at the first methionine residue of the PBOMP-2 signal sequence (i.e. the initiation codon of the PBOMP-2 ORF); and is apparently due to reinitiation of translation at this point; and
  • the lower 15,000 dalton MW band is blocked to N- terminal analysis and can be labeled with 14 C- palmitate. Hence, this band consists of lipoprotein processed PBOMP-2.
  • P1-P5 Five peptides, P1-P5, corresponding to the two end termini were selected for chemical synthesis.
  • Peptide P1 corresponds to the N-terminal residues 1-20 of the mature protein (see FIG. 26 and 27). The remaining four peptides,
  • P2-P5 are a nested series from the C-terminal end of PBOMP-
  • Synthetic peptides were prepared by the solid phase method of
  • Proteolytic fragments of PBOMP-1 were obtained by digesting with a variety of proteases. EndoLys-C digestion resulted in the generation of a large 63 amino acid peptide (residues 1-63) of the mature protein. Tyr 87-Glu 121 was obtained from a digest with Staphylococcus V8 protease and
  • Gly 18-Arg 85 was derived from an endoArg-C digestion.
  • Monoclonal antibodies were produced and purified as described in Section 6.2.2. Briefly, hybridoma cell lines secreting antibodies to PBOMP-1 were obtained by fusion of mouse myeloma cell line, X63. Ag8,6543 with spleen cells obtained from a C57/B1 mouse immunized with PBOMP-1. Desired hybridomas were recloned by limiting dilution and screened for reactivity for PBOMP-1 by Western Blot. Selected
  • hybridomas were injected into Balb/c mice for growth as ascites.
  • a direct ELISA assay was used to determine the binding of Mabs to synthetic peptides of PBOMP-1. As indicated in
  • Mabs G204-2 and G194-3 lies within the first twenty amino acids of PBOMP-1.
  • Mabs G196-3, G212-6, G214-3, and G190-8 to PBOMP-1 was effectively inhibited (100%) by all of the C- rermini peptides, P2-P5 (Table 11) .
  • These four Mabs must be recognizing an epitope(s) contained within the P2 peptide, a 15 amino acid sequence spanning Aspl20-Tyrl34.
  • E-K residues 103-113 of the mature protein
  • PBOMP-1 peptide fragments generated by proteolytic cleavage were tested for reactivity with a limited number of anti-PBOMP-1 Mabs by direct dot blot and Western immunoblot analysis.
  • a large 63 amino acid peptide (residues 1-63 of the mature protein) obtained from endoLys-C digestion, reacted with Mab G204-2. This observation was not surprising since the N-terminal 20 amino acid sequence is contained within this cleavage peptide.
  • Mab G219-3 which did not bind with any of the synthetic peptides, was shown to react with a 68 amino acid peptide, Glyl8-Arg85, derived from an endoArg-C digestion.
  • FIG. 28 A schematic diagram representing the mapping of epitopes recognized by Mabs is depicted in FIG. 28. Two of the four determinants were localized at the chain termini, i.e, the N-terminal residues 1-20 and the C-terminal 120-134 of the mature protein. The third antigenic region, recognized by Mab G216-3 and G187-1, was localized to a small amino acid sequence (10 amino acids) spanning residues 103- 113 of the mature protein. Finally, a rather large antigenic region, recognized by Mab G219-3, is encompassed by residues Glyl8-Arg85. Since the latter peptide is quite large, the epitope could likely be conformation-dependent, since
  • the functional activity of the Mabs were tested in a bactericidal assay against H. influenzae strain S2.
  • the complement used was precollostral calf serum (PCCS) adsorbed 2X with washed S2 whole cells.
  • Bacteria were grown overnight in brain heart infusion (BHI) broth supplemented with hemin at 10 ug/ml and NAD at 2 ug/ml (BHIXV), then diluted 1:15 in fresh broth and incubated at 37oC with aeration. Cells were grown to an OD at 490 nm of 0.9 (approximately 10 CFU/ml).
  • Bacteria were diluted 40,000 fold by a series of dilution in sterile phosphate-buffered saline containing 0.15 mM CaCl 2 ,
  • PCMA bovine serum albumin
  • the peptide selected for synthesis in this study corresponded to the N-terminal 20 amino acid sequence, Cysl-
  • the conjugation process proceeded in a stepwise manner.
  • the epsilon amino groups of surface lysinyl residues of BSA (or thyroglobulin) were N- acylated with an excess of MBS.
  • the coupling reaction was carried out at room temperature in phosphate-buffered saline, pH 7.3, by mixing the protein carrier with MBS at a molar ratio of 1:100. The reaction mixture was desalted over
  • conjugation was achieved by adding the reduced peptide to the solution containing the BSA-MBS or thyroglobulin-MBS derivative and allowing the covalent reaction to take place overnight. Dialysis was performed to remove excess unreacted peptide prior to freeze-drying.
  • molecular weight corresponded to 7 molecules of peptide per
  • N-terminal peptide was fatty-acid acylated with palmityl groups according to the method of Hopp (1984, Mol. Immuno. 21: 13-16). Essentially, the peptide was elongated at the ammo terminus with diglycine spacer followed by a lysyl residue. Both the alpha- and epsilon-amino groups of N-terminal lysine were acylated with palmitic acids by the symmetric anhydride coupling procedure.
  • mice which were at least 6 weeks old were obtained from Taconic Farms (Germantown, NY). Groups of 5 mice were pre-bled and vaccinated intramuscularly with one or 10 ug of a (N-1-20) carrier peptide conjugate, with and without complete Freund's adjuvant (CFA). Animals were boosted at week 4 with the same dose of vaccine used for priming. Mice primed with CFA were boosted with conjugate in incomplete Fueund's adjuvant (IFA). Mice were bled at weeks 4 and 6.
  • CFA complete Freund's adjuvant
  • Hybridoma cell lines secreting antibodies to the
  • PBOMP-1 N-terminal peptide-carrier conjugate were obtained by the fusion of the mouse myeloma cell line, X63.Ag8.6543 with spleen cells obtained from a C57/B1 mouse immunized with (N-
  • Desired hybridomas were recloned by limiting dilution (see Section 6.2.2. for a detailed description of procedure) and screened for reactivity with (N-1-20)-carrier conjugate by
  • Anti-(N-1-20) peptide activity of the animal antisera was determined by the ELISA method as described in Section 6.2.2., supra. Briefly, 96 well polystyrene plates (NUNC Intermed, Thousand Oaks, CA) were coated with PBOMP-1 or a (N-1-20)-peptide carrier conjugate, i.e., (N-1-20)-peptide- BSA, at a concentration of 1 ug/ml in PBS. Plates were incubated overnight at 37° C in humidified chamber. Prevaccination serum was always used as a negative control and a polyclonal anti-PBOMP-1 serum was used as a positive control.
  • Alkaline phosphatase coupled to IgG anti-mouse antisera was used as secondary antibody.
  • the reaction was developed with p-nitrophenylphosphate at 1 mg/ml in diethanolamine buffer.
  • optical density (OD) at 410 and 690 nm was read using a Bio- Tek 310 Autoreader. Results are presented in Table 13.
  • mice immunized with (N-1-20)-carrier conjugate gave a high anti-peptide titer response of 1:1,024,000.
  • the mouse anti-peptide antibodies also strongly recognized the parent PBOMP-1 in the ELISA at a 1:512,000. These antipeptide antibodies appear to be specific to the N-termmal peptide since only negligible amounts of these antibodies reacted with BSA.
  • Rabbit #1 The maximum rabbit anti-peptide titer was obtained with Rabbit #2 at 1:4,096,000. Rabbit #1 also had a strong anti peptide titer of 1:256,000. Both the rabbit anti-peptide sera were capable of recognizing native PBOMP-1 as seen by the 32-fold and 512-fold increase in titers compared to the preimmune titers of Rabbit #1 and Rabbit #2, respectively.
  • mice antipeptide sera raised to the (N-1-20)- carrier conjugate were evaluated.
  • the antipeptide antibody and the mouse anti-PBOMP-1 polyclonal sera were observed to react with both conjugates: peptide-BSA and peptide-thyroglobulin, and also with native PBOMP-1 on immunoblots.
  • the Western blot results also revealed that little, if any, antibodies were made to the MBS-spacer since the antipeptide sera did not react with a control conjugate consisting of 'nonsense' peptide (of an equivalent size) linked to BSA through the MBS spacer.
  • the palmityl-peptide conjugate was also not immunogenic in mice.
  • the N-terminal peptide was coupled to a protein carrier, e.g., thyroglobulin.
  • the bactericidal activity (BC) of mouse anti-(N-1-20) peptide antisera against H. influenzae S2 strain was measured using procedures described in Section 9.4, supra.
  • mice immunized with free peptide were capable of killing S2 organisms at a dilution of 1/40, compared to mouse anti-PBOMP-1 polyclonal sera at 1/ 80.
  • mice immunized with free peptide were immunized with free peptide
  • rabbit #2 antisera which had the higher anti-(N-1-20) titer, to have the higher BC activity; however, rabbit #1 antiserum exhibits better killing effect.
  • FIG. 30 diagramatically in FIG. 30.
  • DNA fragments including the PBOMP-1 and PBOMP-2 genes were derived from plasmids pPX167 and pPX163 respectively. The isolation of each of these plasmids is described in
  • Plasmid pPX183 was transformed into E. coli JM103 and the resulting strain was tested for production of a
  • PBOMP-2 PBOMP-1 FUSION PROTEIN
  • plasmid pPX183 encodes a hybrid PBOMP-1/
  • PBOMP-2 protein i.e. a fusion protein
  • the protein expressed by this plasmid contains additional amino acids encoded by the pUC19 vector.
  • the nucleotide sequence of pPX183 contains 18 additional codons at the 5' end from pPX163 and 13 additional codons at the fusion junction - 31codons derived from the pUC19 vector.
  • a new fusion gene lacking most of this excess information was constructed as follows.
  • pAA130 containing the PBOMP-2 gene was digested with Sspl, which cleaves 5 nucleotides 5'to the initiation codon of PBOMP-2, and then with Hindlll which cleaves the PBOMP-2 gene at codon 148, i.e., 6 codons from the 3' end of the gene. Sequence analysis showed that this fragment could be ligated into plasmid pUC8 and digested with Smal and Hindlll to generate a recombinant PBOMP-2 gene with seven codons fused to the 5' end of the "native" gene and six amino acids missing at the 3' end. A plasmid was produced in this manner and designated pPX195 (see FIG. 31).
  • the BamHl site at th 3' end of the gene was removed by partial digestion with BamHl and religated by treatment with
  • pX188 E. coli DNA polymerase I (Klenow fragment).
  • the resulting plasmid designated pX188 was verified by restriction analysis and production of rPBOMP-1 (see FIG. 31) .
  • the 5 ' end of the rPBOMP-1 gene of pPX188 was modified by removal of the
  • Plasmids pPX195 and pPX198 were digested with Hindlll and Seal and the fragments carrying the PBOMP-2 and PBOMP-1 genes respectively were isolated and ligated together. The resulting plasmid was designated pX199 (FIG. 31). Plasmid pX199 expresses a PBOMP-2: PBOMP-1 fusion gene with seven additional codons (from pUC8) at the 5' end and two at the fusion junction. The fusion protein has an apparent
  • PBOMP-2 PBOMP-1 fusion protein gene of pPX199 encodes the PBOMP-2 signal peptide, and the resulting fusion protein is modified as a lipoprotein and tightly associated with the outer membrane of E. coli cells expressing it. This fusion protein is difficult to separate from the membrane and poorly soluble in aqueous solvents. Construction of a plasmid which expresses a signal-less PBOMP-2: PBOMP-1 fusion protein is illustrated in FIG. 33.
  • a signal-less form of the PBOMP-2 gene was constructed in the following manner: The 660 bp EcoRI/Sall restriction fragment of plasmid pPX163 encoding the PBOMP-2 protein was isolated and cleaved at the unique Hphl site at nucleotide 91 of the gene and the 545 bp Hphl/EcoRI fragment encoding the carboxyterminal 125 amino acids of PBOMP-2 was isolated.
  • the linker was designed to create a modified PBOMP-2 gene with the following features:
  • SD box A new translation initiation site (SD box), and (6) An upstream in frame stop codon (TAA) to prevent lac translational readthrough.
  • TAA upstream in frame stop codon
  • the linker was mixed at a 1:1:1 molar ratio with the
  • Plasmid pPX510 encodes a 14000 apparent dalton
  • a signal-less fusion gene was then constructed from pPX510 and pPX198 in a manner analogous to the construction of pPX199 (see Section 11.3, supra). Briefly, the two plasmids were digested with Hindlll and Seal and the PBOMP-1 and PBOMP-2 fragments were ligated together. The resulting plasmid was transformed into E. coli DH 5 ⁇ (BRL,
  • pPX512 The signal-less fusion protein expressed by pPX512 had an apparent molecular weight of 30000 daltons by SDS-PAGE and was recognized by polyclonal antisera against both PBOMP-1 and PBOMP-2. The fusion protein was also recognized by PBOMP-2 MAb 61-1 and all PBOMP-1 Mabs tested by Western blot analysis.
  • PBOMP-1 PBOMP-1 ANTIBODIES
  • PBOMP-1 was isolated from an SDS-PAGE gel of cell extracts of E. coli JM103 containing pPX183 (see Section 11.1, supra). The position of the fusion protein band on the preparative SDS-PAGE gel was deduced by
  • PBOMP-2 PBOMP-1 elicited antibody responses to both PBOMP-1 and PBOMP-2 as determined in ELISA assays and these antisera had bactericidal activity against the unencapsulated H.
  • PBOMP-2 PBOMP-1 fusion protein is highly effective in provoking an antibody response against both PBOMP-1 and
  • chinchillas were used as a test system to demonstrate further the immunogenicity and efficacy of the PBOMP-1: PBOMP-2 fusion protein as a subunit vaccine formulation.
  • the chinchilla serves as a model animal system because this animal when infected with H. influenzae develops an otitis media much like that seen mr humans. (See, Barenkamp et al., 1986,
  • the PBOMP-2 PBOMP-1 fusion protein expressed by E . coli JM103 containing pPX199 is modified as a lipoprotein and is tightly associated with the outer membrane of the E. coli host cells.
  • Substantially pure PBOMP-2 PBOMP-1 was isolated from E. coli JM103 containing pPX199 by the following method entailing
  • PBOMP-1 fusion protein was obtained by differential detergent extraction of other outer membrane-cell wall components.
  • the outer membrane proteins were extracted from the sarcosyl insoluble outer membrane cell wall components by sequential extraction with 1% Zwittergent 3-12TM, 1% Zwittergent 3-14 TM 10 mM
  • the PBOMP-2 PBOMP-1 fusion protein enriched insoluble material was obtained following centrifugation.
  • the PBOMP-2 PBOMP-1 fusion protein was solubilized by extraction of the PBOMP-2:
  • the apparent purity of the solubilized fusion protein was in the range of 75-80%. Approximately 25 mg of protein was solubilized from ⁇ 10-20 g wet weight fusion protein. The major protein was identified as the intact fusion protein.
  • the major lower MW protein also was derived from the fusion protein since it contained the N-terminal region of the
  • PBOMP-1 protein fused to the PBOMP-2 protein as determined by epitope analysis.
  • the purity of this preparation is shown in FIG. 32.
  • PBOMP-2 PBOMP-1 fusion protein, obtained from E. coli cells containing plasmid pPX199 as described above, absorbed onto alum was injected intramuscularly into the chinchillas.
  • PBOMP-2 PBOMP-1 FUSION PROTEIN IN COMBINATION WITH
  • the E protein is another outer membrane protein of H. influenzae having a molecular weight of about 28,000 daltons. E protein exists as a lipoprotein in association with other outer membrane proteins of H. influenzae.
  • E protein from H. influenzae was obtained in
  • cell envelopes were isolated from Hib strain Eagan cells grown on either brain heart infusion medium containing io ⁇ g/ml hemin and 1 ⁇ g/ml NAD (BHI/XV) or mMIC
  • An outer membrane-cell wall complex was obtained by removing the inner membranes from the total membrane fraction by repeated extraction of the total membrane fraction with 2% Triton X-100 in 10 mM HEPES-NaOH, 1 mM MgCl 2 , pH.7.4. The insoluble residue containing the outer membrane-cell wall complex was pelleted by centrifugation at 350,000 x g for 30 minutes at 4°C. This complex was then resuspended in 50 mM
  • influenzae cell envelopes by differential detergent
  • the precipitated E protein was then solubilized again by differential detergent extraction.
  • the precipitate was first extracted with 1% octylglucoside in 50 mM Tris-HCl, pH 8 and the insoluble E protein remained in the precipitate.
  • the protein E was then solubilized with 1% Zwittergent 3-14TM in
  • E Protein prepared as desribed above is substantially pure and essentially free of endotoxin.
  • PBOMP-1 fusion protein obtained as described in Section 12.1, supra, diluted into 0.85% saline and absorbed with alum.

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US5300632A (en) * 1986-11-18 1994-04-05 Research Foundation Of State University Of New York Method for purifying an outer membrane protein of Haemophilus influenzae
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US5679547A (en) * 1990-12-21 1997-10-21 Antex Biologics Formerlly Microcarb Inc. Method for producing a novel purified Haemophilus influenzae protein
US5679352A (en) * 1992-02-03 1997-10-21 Connaught Laboratories Limited Synthetic Haemophilus influenzae conjugate vaccine
WO1993015205A2 (en) * 1992-02-03 1993-08-05 Connaught Laboratories Limited Synthetic haemophilus influenzae conjugate vaccine
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AU4228889A (en) 1990-04-02
AU651030B2 (en) 1994-07-07
DK35891D0 (da) 1991-02-28
JPH04502147A (ja) 1992-04-16
EP0432220A4 (en) 1991-12-04
EP0432220A1 (de) 1991-06-19
DK174965B1 (da) 2004-04-05
DE68923286D1 (de) 1995-08-03
ATE124420T1 (de) 1995-07-15
AU3379693A (en) 1993-04-29
DK35891A (da) 1991-04-30
KR0162488B1 (ko) 1998-11-16
JP3073212B2 (ja) 2000-08-07
AU631378B2 (en) 1992-11-26
DE68923286T2 (de) 1996-03-07
KR0170752B1 (ko) 1999-10-01
KR900701291A (ko) 1990-12-01
EP0432220B1 (de) 1995-06-28
CA1340888C (en) 2000-02-01

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